Antitumor cytotoxic cell activity generated in vitro by normal mouse spleen cells under fully syngeneic conditions has been further investigated. Generation of cytotoxic cells, the specificity of which is not H-2 restricted and includes some types of normal cells lines, is under genetic control which is not part of the major histocompatibility complex. To date only mice descended from C57BL are responders and responsiveness is dominant or codominant. Responsiveness is not regulated by the source of mouse serum used in the culture medium.